Unit: DNA Micro-arrays (Platform)
Director: Coppée Jean-Yves
The aim of the platform is to develop biochips technology for the study (comparative genomics and gene expression profiling) of model microorganisms and infectious diseases (pathogen microorganisms and response of the host to the infection).
We have the equipment for making and using DNA chips on various supports (glass slides, nylon membranes, Affymetrix chips) . We are also developing the bioinformatical and statistical tools necessary for the analysis, interpretation and handling of the results.
The availability of the complete genome sequences of a growing number of microorganisms and the emergence of new technologies for the study of gene expression lead the Pasteur Institute to set up a platform dedicated to the fabrication and analysis of biochips for the study of model microorganisms and infectious diseases. These studies involve both the agents responsible for these pathologies (comparative genomics and gene expression profiling) and the response of the host (gene expression profiling).
At present, three technologies are used at the platform :
Microarrays : spotting of PCR products or long oligos onto glass slides and hybridization with fluorescent probes
Macroarrays : spotting of PCR products onto nylon membranes and hybridization with radioactive probes
Affymetrix : use of commercial chips containing in situ synthesized short oligos
The platform is in charge of projects selected by internal calls for proposals. The projects are made as collaborations. Most of the selected projects have national or european frameworks. The costs of fabrication and use of the chips are supported (partially or totally) by the Genopole.
For the selected projects, the platfom is in charge of :
- the design of the oligos (primers for PCR amplification and long oligos for spotting; the long oligos are syntheiszed and quality controlled by PF7)
- the PCR amplifications (including quantification and quality control)
- the spotting onto the nylon membranes or the glass slides
The platform also has training activities for :
- the target preparation (fluorescent and radioactive) and the hybridizations
- the detection and quantification of the hybridization signals (GenePix and Arrayvision softwares)
- the analysis of the results: several tools for the normalization of the data and for their statistical analysis are installed at the platform. We are using mainly the R environment (Bioconductor) as well as commercial softwares (ArrayMiner, GeneSpring, S-Plus-Array-Analyzer). The data analysis is systematically performed with the users.
- the handling of the data: In collaboration with S. Moreira and I. Moszer (Plate-forme Integration et Analyse Genomique), a database has been build according to the MIAME recommendations for the storage of microarrays and macroarrays data.
Pojects centered on pathogens
In 2004, three new projects aiming at gene expression profiling of pathogen microorganisms have been started:
Analysis of the transcriptome of the pathogen fungus Aspergillus fumigatus
A. Beauvais, I. Mouyna and J.P. Latgé (Unité des Aspergillus)
Gene expression profiling in Bacillus anthracis in host's infection like conditions
O. Mary-Possot, M. Mock (Unité des Toxines et Pathogénie Bactérienne)
Functional genomics of Legionella pneumophila for the study of the host-pathogen interactions
C. Buchrieser and P. Glaser (Unité de Génomique des Microorganismes Pathogènes)
Furthermore, the platform keeps on working on projects selected during previous calls for proposals. These projects concern in particular the complete genomes of Candida glabrata (coll. B. Dujon), Plasmodium falciparum (coll. P. David), Yersinia (coll. E. Carniel). Dedicated chips with selected genes from Entamoeba histolytica (coll. N. Guillen) have also been produced. Theses projects aim at the analysis of the modifications of the transcriptome following various environmental or immune pressures, at the comparisons of the transcriptomes of several species from the same genus, at the identifcation of virulence factors and of targets from regulators...
In 2004 we have also set up a platform which is specifically dedicated to comparative genomics. This platform develops chips for the typing and identification of microorganisms and for the study of biodiversity and Public Health problems. It is accessible to all the french National Reference Centers via a call for proposals which is open all year (the projects are selected by a steering comittee). Two projects have been started in 2004: one project aims at the genomic typing of Legionella pneumophila (coll. C. Buchrieser) and another project at the molecular idenfication of species from the Bordetella genus (coll. N. Guiso). These projects received fundings for the making and the use of the chips.
Projects centered on higher eukaryotes
In 2003, the genopole has acquired an Affymetrix station (including an hybridization oven, fluidic station, a scanner and softwares for the analyses of hybridization images and results) for studying gene expression profiles in higher eukaryotes and in particular for biological questions centered on development or on the response of the host to the infection. The platform is in charge of the quality control of the RNAs, their labeling, the hybridization, the analysis of the images and of the results (this step is performed in close collaboration with the biologists).The projects are selected following calls for proposals (open all year) and receive fundings (2/3 of the cost of the project being supported by the Pasteur Institute).
In 2004, the platform has been in charge of 8 projects:
Response of human cells to a mycobacterial toxin: mycolactone.
C. Demangel, E. Coutanceau, L. Marsollier and S.T. Cole; Bacterial Molecular Genetics Unit
Analysis of the transcriptome of xenografts during Shigella flexneri infection.
T. Pédron, C. Parsot and P. Sansonetti; Molecular Microbial Pathogenesis Unit, Inserm U389
Identification of genes involved in the morphogenesis of olfactory bulbs and in cellular migration.
N.Soussi-Yanicostas ; Sensory Deficit Genetics Unit
Determination of the transcriptome of myosatellite cells at rest and after activation
M. Buckingham ; Molecular Genetics of Development Laboratory
Identification of the targets of Pax3
M. Buckingham ; Molecular Genetics of Development Laboratory
Analysis of the transcriptome of primo-cultures of astrocytaire and microgliale cells after interaction with CD8 T lymphocytes of mouse brains which have developped neuro-malaria.
S. Pied ; Immunophysiopathology of Infections Unit
In vivo study of the transcriptome of the murine respiratory apparatus during infection by the pulmonary pathogens Klebsiella pneumoniae and Streptococcus pneumoniae (part two).
R. Tournebize, T. Pédron, and P. Sansonetti ; Molecular Microbial Pathogenesis Unit, Inserm U389
Study of the transcriptional modifications during rabid encephalitis in humans and dogs using DNA micro arrays.
H. Bourhy ; Rabies Laboratory
Keywords: Transcriptomics, comparative genomics, infectious diseases, macroarrays, microarrays, Affymetrix